Worldwide, liver diseases such as hepatitis, both viral and non-viral induced, are the primary causes of gastrointestinal (GI) disorders affecting hundreds of thousands of people (1-3). Hepatitis invariably leads to liver fibrosis, which may have drastic effects. Hepatitis virus infections including the hepatitis B virus (HBV) and the hepatitis C virus (HCV) are currently the major causes of liver diseases, which are endemic in many regions of the world (4-14). Many regions in the world, including the Western Pacific Region such as China, Taiwan, Hong Kong and Southeast Asia, are major endemic areas for hepatitis virus infections, which are mainly HBV and HCV (4-14).
In addition, chronic alcohol consumption, metabolic and autoimmune disorders, toxic/drug-induced injuries and biliary obstruction can also produce hepatitis and hepatic fibrosis. If liver fibrosis is left untreated, it can progress to overt liver cirrhosis and hepatic failure. Liver cirrhosis can also progress to primary hepatocellular carcinoma, which is one of the major malignancies in Africa and Asia, including China, Taiwan and Hong Kong. It is not surprising that liver disease is currently the 11th leading cause of death in the world (11-12). Furthermore, the rates of alcohol and drug abuses, which often leading to liver inflammation, in many immerging industrialized region are on the rise.
Liver fibrosis is formed as a consequence of continuous damage to the liver, caused by alcohol abuse, autoimmune diseases, drugs, metabolic diseases and viral hepatitis. The disease is characterized by excessive production and accumulation of extracellular matrix (ECM) proteins, which includes collagen types I, II, IV, V, VI and VII, fibronectin, undulin, elastin, laminin, hyaluronan and proteoglycans etc. The end stage of liver fibrosis is cirrhosis, which is characterized by: (a) extensive scar tissue formations, (b) the appearance of regenerative nodules accompanied by liver failure and (c) portal hypertension.
Liver fibrosis represents a tremendous worldwide healthcare problem. The biomarkers for liver fibrosis and hepatitis have been documented previously (8,9). Knowledge of the cellular and molecular mechanisms of liver fibrosis has now greatly advanced. Activated hepatic stellate cells (HSC), portal fibroblasts, and myofibroblasts of bone marrow origin have been identified as the major collagen-producing cells in the diseased liver (5,6). These cells are activated by fibrogenic cytokines such as TGF-beta1, angiotensin II, and leptin.
Recently, it has been reported that liver fibrosis could be reversed in patients, thus stimulating researchers to develop antifibrotic drugs. Emerging antifibrotic therapies are aimed at inhibiting the accumulation of fibrogenic cells and/or preventing the deposition of extracellular matrix proteins.
Therefore, it is important that drugs are developed specifically to target ECM producing cells such as activated HSC to combat liver fibrosis. In China, it has been estimated that the market value for medicines of liver disease is approximately 10 billion RMB, of which patented medicines in China account for 2 billion RMB. Statistics released by the Ministry of Chinese Public Health showed that the incidence of viral hepatitis including HBV and HCV in China is still very high and continues to rise. In 2010, the reported new cases for viral hepatitis in China were more than 1.3 million (13). There are approximately 120 million hepatitis sufferers in China that support the huge market of liver-disease medication.
At present, medicines for liver disease can be divided into several approaches. The main focus is on anti-viral medicines such as interferon, and ribavirin, a synthetic nucleoside analog that inhibits the viral genome duplication. In addition, lamivudine has been used for chronic hepatitis B infection, while treatment of hepatitis C and hepatitis D is with interferon and treatment of primary biliary cirrhosis is with methotrexate plus ursodiol.
According to the National Institutes of Health (NIH) of Bethesda, Md., USA, current treatments for HBV are interferons (interferon-α2b and peginterferon-α2a) and/or nucleoside or nucleotide analogues (lamivudine, adefovir, entecavir, Tenofovir Disoproxil Fumarate (TDF), telbivudine, Emtricitabine plus TDF and Truvada, (TVD) (8). The Current treatment for HCV infection is usually Peginterferon (PEG-IFN) Alfa-2b plus Ribavirin (RBV) (9).
However, the long-term usage of many of these drugs for HBV and HCV may develop drug-resistance, which is not beneficial to patients of chronic diseases. The administration of these drugs also requires long period of hospitalization and closed monitoring, which are costly and inconvenient. The world market for interferons alone was valued at US$3.8 billion in 2001. However, the cost of these treatments is enormously high and the effects are far from satisfactory. In addition, many of these patients are not eligible for liver transplant, and thus the prognosis for them is terribly ominous.
Recent research and developments have changed the dynamics of the treatment approach, and opened demands for medicine involved in reversing liver fibrosis, liver immunity regulation, and liver protection. It is interesting to note that one of the direct ways to treat liver disease and liver fibrosis is to inhibit the formation and the deposition of collagens and ECM. However, there is no product currently available that are effective in targeting the expression of collagens, ECM and limiting liver fibrosis. Thus, novel new drugs and methodologies are urgently needed to alleviate pains and sufferings of patients with hepatitis and liver fibrosis.
Hesperadin, an Aurora Kinase Inhibitor
To combat cancer and proliferative diseases, aurora kinase inhibitors are considered to be extremely useful because aurora kinases were found to be over-expressed in many types of cancer, and they could be responsible for the high proliferation and the loss of growth control in tumors (28-32). Small molecules that are aurora kinase inhibitors, such as Hesperadin, VX680 and reversine are good candidates for anti-cancer applications since they can be chemically produced, and are relatively stable, penetrable and permeable to various tissues and cells (28-42).
Hesperadin is a human Aurora B kinase inhibitor with a half maximal inhibitory concentration (“IC50”) of 40 nM for the prevention of the phosphorylation of substrate (40-55). Hesperadin blocks nuclear division and cytokinesis, but not other aspects of the cell cycle (40). Mammalian cells treated with Hesperadin enter anaphase in the presence of numerous mono-oriented chromosomes, many of which may have both sister kinetochores attached to one spindle pole (syntelic attachment) (40,45). Hesperadin also induced cells arrested by taxol or monastrol to enter anaphase within <1 h, whereas cells in nocodazole stayed arrested for 3-5 h. Moreover, the proper segregation of sister chromatids in mitosis depends on the bipolar attachment of all chromosomes to the mitotic spindle. Thus, Hesperadin has been identified as an effective inhibitor of chromosome alignment and segregation. Growth of cultured bloodstream forms (BF) was also sensitive to Hesperadin with an IC50 of 50 nM (41).
These data implied that Hesperadin causes this phenotype by inhibiting the function of the mitotic kinase Aurora B (40). It suggested that Aurora B is required to generate unattached kinetochores on mono-oriented chromosomes, which in turn could promote bipolar attachment as well as maintain checkpoint signaling. Since the expression of aurora kinases were found to be up-regulated in many types of cancer, inhibitors of aurora kinases are thought to be extremely useful in combating neoplasia, proliferative and fibrotic diseases (28-32). Nevertheless, Hesperadin has not been documented for inhibiting liver fibrosis and hepatitis.

Moreover, Publication WO 2002/036564 A1 (Publication '564) of International Patent Application PCT/EP2001/012523 describes indolinone derivatives including Hesperadin that are directed to the inhibition of the proliferation of tumor cells and protein kinases (60). However, Publication '564 does not describe the use of Hesperadin and related indolinones therein as inhibitors of liver fibrosis or inflammation.
Indolinone Derivatives for Fibrotic Diseases
Several U.S. patent application publications have reported using indolinone derivatives to treat fibrotic diseases (55-59). For instance, U.S. Patent Publications 2006/0142373 A1 (Publication '373), 2006/0148883 A1 (Publication '883), and 2006/0154939 A1 (Publication '939), utilize indolinones that can be represented by the following general formula for fibrotic diseases:
wherein R1, R2, R3, R4, R5 and R6 are defined. Because the substitution at position 5 of the indolinone is R6, which is hydrogen, the indolinones in Publications '373, '883 and '939 are structurally different and not related to Hesperadin. In addition, Publications '373, '883 and '939 do not describe the indolinones therein as biologically active against hepatic stellate cells (HSC).
On the other hand, U.S. Patent Publications 2009/0048267 A1 (Publication '267) and 2010/0204211 A1 (Publication '211) also utilize indolinones that can be represented by the following general formula for fibrotic diseases:
wherein R1, R2, R3, R4, and R5 are defined. Because position 5 of the indolinone is hydrogen, the indolinones in Publications '267 and '211 are structurally different and not related to Hesperadin. Furthermore, Publications '267 and '211 only presented biological activities related to bleomycin-induced lung fibrosis, and Publication '267 also describes virally induced hepatic cirrhosis. However, Publications '267 and '211 do not describe the indolinones therein as biologically active against hepatic stellate cells (HSC).
Therefore, there remains a need to utilize indolinones that are structurally related to Hesperadin but are capable of inhibiting hepatic stellate cells (HSC) as treatments of liver fibrosis and hepatitis.